Human proteinase molecules

ABSTRACT

The invention provides human proteinase molecules, the polynucleotides which encode them, and methods for their use. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention further provides methods for diagnosing or treating disorders associated with expression of HPRM.

[0001] This application is a DIVISIONAL of pending prior applicationU.S. Ser. No. 09/802,633, filed on Mar. 8, 2001, which in turn is aDIVISIONAL of prior application U.S. Ser. No. 09/032,523, filed on Feb.27, 1998, now issued as U.S. Pat. No. 6,232,454 B1 on May 15, 2001, bothentitled HUMAN PROTEINEASE MOLECULES, both of which are hereby expresslyincorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof human proteinase molecules and to the use of these sequences in thediagnosis and treatment of cancer and immune disorders.

BACKGROUND OF THE INVENTION

[0003] Proteolytic processing is an essential component of normal cellgrowth, differentiation, remodeling, and homeostasis. The cleavage ofpeptide bonds within cells is necessary for the maturation of precursorproteins to their active form, the removal of signal sequences fromtargeted proteins, the degradation of incorrectly folded proteins, andthe controlled turnover of peptides within the cell. Proteasesparticipate in apoptosis, antigen presentation, inflammation, tissueremodeling during embryonic development, wound healing, and normalgrowth. They are necessary components of bacterial, parasitic, and viralinvasion and replication within a host. Four principal categories ofmammalian proteases have been identified based on active site structure,mechanism of action, and overall three-dimensional structure (Beynon andBond (1994) Proteolytic Enzymes: A Practical Approach, Oxford UniversityPress, New York N.Y., pp. 1-5).

[0004] The serine proteases (SPs) are a large family of proteolyticenzymes that include the digestive enzymes, trypsin and chymotrypsin;components of the complement cascade and of the blood-clotting cascade;and enzymes that control the degradation and turnover of macromoleculesof the extracellular matrix. SPs are so named because of the presence ofa serine residue found in the active catalytic site for proteincleavage. The active site of all SP is composed of a triad of residuesincluding the aforementioned serine, an aspartate, and a histidineresidue. SPs have a wide range of substrate specificities and can besubdivided into subfamilies on the basis of these specificities. Themain sub-families are trypases which cleave after arginine or lysine;aspases which cleave after aspartate; chymases which cleave afterphenylalanine or leucine; metases which cleavage after methionine; andserases which cleave after serine.

[0005] The SPs are secretory proteins containing N-terminal signalpeptides which export the immature protein across the endoplasmicreticulum prior to cleavage (von Heijne (1986) Nuc Acid Res14:5683-5690). Differences in these signal sequences provide one meansof distinguishing individual SPs. Some SPs, particularly the digestiveenzymes, exist as inactive precursors or preproenzymes and contain aleader or activation peptide on the C-terminal side of the signalpeptide. This activation peptide may be 2-12 amino acids in length andextend from the cleavage site of the signal peptide to the N-terminus ofthe active, mature protein. Cleavage of this sequence activates theenzyme. This sequence varies in different SPs according to thebiochemical pathway and/or its substrate (Zunino et al. (1990) J Immunol144:2001-2009; Sayers et al. (1994) J Immunol 152:2289-2297).

[0006] Cysteine proteases are involved in diverse cellular processesranging from the processing of precursor proteins to intracellulardegradation. Mammalian cysteine proteases include lysosomal cathepsinsand cytosolic calcium activated proteases, calpains. Cysteine proteasesare produced by monocytes, macrophages and other cells of the immunesystem which migrate to sites of inflammation and in their protectiverole secrete various molecules to repair damaged tissue. These cells mayoverproduce the same molecules and cause tissue destruction in certaindisorders. In autoimmune diseases such as rheumatoid arthritis, thesecretion of the cysteine protease, cathepsin C, degrades collagen,laminin, elastin and other structural proteins found in theextracellular matrix of bones. The cathepsin family of lysosomalproteases includes the cysteine proteases; cathepsins B, H, K, L, O2,and S; and the aspartyl proteases; cathepsins D and E. Various membersof this endosomal protease family are differentially expressed. Some,such as cathepsin D, have a ubiquitous tissue distribution while others,such as cathepsin L, are found only in monocytes, macrophages, and othercells of the immune system.

[0007] Abnormal regulation and expression of cathepsins has beenimplicated in various inflammatory disease states. In cells isolatedfrom inflamed synovia, the mRNA for stromelysin, cytokines, TIMP-1,cathepsin, gelatinase, and other molecules is preferentially expressed.Expression of cathepsins L and D is elevated in synovial tissues frompatients with rheumatoid arthritis and osteoarthritis. Cathepsin Lexpression may also contribute to the influx of mononuclear cells whichexacerbate the destruction of the rheumatoid synovium (Keyszer (1995)Arthritis Rheum 38:976-984). The increased expression and differentialregulation of the cathepsins is linked to the metastatic potential of avariety of cancers and may be of therapeutic and prognostic interest(Chambers et al. (1993) Crit Rev Oncog 4:95-114).

[0008] Cysteine proteases are characterized by a catalytic domaincontaining a triad of amino acid residues similar to that found inserine proteases. A cysteine replaces the active serine residue.Catalysis proceeds via a thiol ester intermediate and is facilitated bythe side chains of the adjacent histidine and aspartate residues.

[0009] Aspartic proteases include bacterial penicillopepsin, mammalianpepsin, renin, chymosin, cathepsins D and E, and certain fungalproteases. The characteristic active site residues of aspartic proteasesare a pair of aspartic acid residues, such as asp33 and asp213 inpenicillopepsin. Aspartic proteases are also called acid proteasesbecause the optimum pH for activity is between 2 and 3. In this pHrange, only one of the aspartate residues is ionized. A potent inhibitorof aspartic proteases is the hexapeptide, pepstatin, which in thetransition state resembles a normal substrate of the enzyme.

[0010] Metalloproteases use zinc as an active site component and aremost notably represented in mammals by the exopeptidases,carboxypeptidase A and B, and the matrix metalloproteases, collagenase,gelatinase, and stromelysin. Carboxypeptidases A and B are exopeptidasesof similar structure and active sites. Carboxypeptidase A, likechymotrypsin, prefers hydrophobic C-terminal aromatic and aliphatic sidechains, whereas carboxypeptidase B is directed toward basic arginine andlysine residues. The matrix-metalloproteases are secreted by connectivetissue cells and play an important role in the maintenance and functionof the basement membrane and extracellular matrix. A naturally occurringinhibitor of metalloproteases, tissue inhibitor of metalloproteases(TIMP), has been shown to prevent the invasion of tumor cells throughbasement membrane, n vitro, indicating the importance of these enzymesin cell invasion processes such as tumor metastasis and the inflammatoryresponse (Mignatti et al. (1986) Cell 47:487-498).

[0011] Protease inhibitors play a major role in the regulation of theactivity and effect of proteases. They have been shown to controlpathogenesis in animal models of proteolytic disorders (Murphy (1991)Agents Actions Suppl 35:69-76). In particular, low levels of thecystatins, low molecular weight inhibitors of the cysteine proteases,seem to be correlated with malignant progression of tumors (Calkins etal. (1995) Biol Biochem Hoppe Seyler 376:71-80). The balance betweenlevels of cysteine proteases and their inhibitors is also significant inthe development of disorders. Specifically, increases in cysteineprotease levels, when accompanied by reductions in inhibitor activity,are correlated with increased malignant properties of tumor cells andthe pathology of arthritis and immunological diseases in humans.

[0012] The discovery of new human proteinase molecules and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis and treatment ofcancer and immune disorders.

SUMMARY OF THE INVENTION

[0013] The invention features purified polypeptides, human proteinasemolecules, referred to collectively as “HPRM” and individually as“HPRM-1”, “HPRM-2”, and “HPRM-3”. In one embodiment, the purifiedpolypeptide, HPRM, comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, andfragments of SEQ ID NOs:1-3. The invention includes a purified varianthaving at least 90% amino acid identity to the amino acid sequences ofSEQ ID NOs:1-3 or fragments thereof.

[0014] The invention provides an isolated and purified polynucleotideencoding the polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:1-3 and fragments thereof. Theinvention also includes an isolated variant having at least 90% sequenceidentity to the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:1-3and fragments thereof.

[0015] The invention also provides an isolated polynucleotide comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6 and fragments and complements of SEQID NOs:4-6. The invention includes a variant having at least 90%sequence identity to the polynucleotide selected from the groupconsisting of SEQ ID NOs:4-6 and complements and fragments thereof.

[0016] The invention further provides an expression vector containing atleast a fragment of the polynucleotide encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-3 and fragments thereof. In another aspect, the expressionvector is contained within a host cell. The invention still furtherprovides a the method for using a polynulceotide to produce apolypeptide comprising culturing the host cell containing an expressionvector containing at least a fragment of a polynucleotide encoding thepolypeptide under conditions for the expression of the polypeptide andrecovering the polypeptide from the host cell culture.

[0017] The invention yet still further provides a method for using apolynucleotide to detect a nucleic acid encoding a polypeptide havingthe amino acid sequence of SEQ ID NOs:1-3 in a sample comprisinghybridizing the polynucleotide or the complement thereof to at least onenucleic acid in the sample, thereby forming a hybridization complex anddetecting the hybridization complex, wherein the presence of thehybridization complex indicates the expression of the nucleic acid inthe sample. In one aspect, the nucleic acids of the sample are amplifiedprior to hybridization.

[0018] The invention additionally provides a method of using apolynucleotide to screen a plurality of molecules to identify a moleculewhich specifically binds the polynucleotide comprising combining thepolynucleotide with the plurality of molecules under conditions to allowspecific binding and detecting specific binding, thereby identifying amolecule which specifically binds the polynucleotide. In one aspect, themolecule is selected from DNA molecules, RNA molecules, peptide nucleicacids, artificial chromosome constructions, peptides, and proteins.

[0019] The method provides purified polypeptides comprising an aminoacid sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, andfragments thereof. In one aspect, a biologically active fragment of thepolypeptide is selected from residues D132-F144 of SEQ ID NO: 1,residues C34-C415 of SEQ ID NO:2 and residues V93-V104 of SEQ ID NO:3.

[0020] The invention also provides a method for using a polypeptide toscreen a plurality of molecules to identify a molecule whichspecifically binds the polypeptide comprising combining the polypeptidewith the plurality of molecules under conditions to allow specificbinding and detecting specific binding, thereby identifying a moleculewhich specifically binds the polypeptide. In one aspect, the moleculesare selected from agonists, antagonists, antibodies, DNA molecules, RNAmolecules, peptide nucleic acids, immunoglobulins, inhibitors, drugcompounds, peptides, and pharmaceutical agents.

[0021] The invention further provides a method of using a polypeptide topurify a molecule which specifically binds the polypeptide from a samplecomprising combining the polypeptide with a sample under conditions toallow specific binding, recovering the bound polypeptide, and separatingthe molecule from the polypeptide, thereby obtaining the purifiedmolecule.

[0022] The invention still further provides a method for using apolypeptide to produce an antibody, comprising immunizing an animal withthe polypeptide under conditions to elicit an antibody response andisolating antibodies which bind specifically to the polypeptide.

[0023] The invention yet further provides a method for using apolypeptide to purify an antibody which specifically binds thepolypeptide comprising combining the polypeptide with a plurality ofantibodies under conditions allow specific binding, recovering the boundpolypeptide, and separating the antibody from the polypeptide, therebyobtaining antibody which specifically binds the polypeptide. In oneaspect, the antibodies are selected from polyclonal antibodies,monoclonal antibodies, chimeric antibodies, single chain antibodies; Fabfragments, Fv fragments, and F(ab′)₂ fragments.

[0024] The invention additionally provides a purified antibody whichspecifically binds the polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-3 and fragmentsthereof.

[0025] The invention provides compositions comprising an isolatedpolynucleotide encoding a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs:1-3 and fragmentsthereof and reporter molecule or a purified polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NOs:1-3 andfragments thereof and a pharmaceutical carrier.

[0026] The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of the polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNOs:1-3 and fragments thereof.

[0027] The invention further provides a method for treating orpreventing an immune disorder, the method comprising administering to asubject in need of such treatment an effective amount of an antagonistof the polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs:1-3 and fragments thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:4) of HPRM-1. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering, SanBruno Calif.).

[0029]FIGS. 2A, 2B, 2C, 2D, and 2E show the amino acid sequence (SEQ IDNO:2) and nucleic acid sequence (SEQ ID NO:5) of HPRM-2. The alignmentwas produced using MACDNASIS PRO software.

[0030]FIGS. 3A, 3B, 3C, 3D, and 3E show the amino acid sequence (SEQ IDNO:3) and nucleic acid sequence (SEQ ID NO:6) of HPRM-3. The alignmentwas produced using MACDNASIS PRO software.

[0031]FIGS. 4A and 4B show the amino acid sequence alignments betweenHPRM-1 (456885; SEQ ID NO:1), and a pig calpain I light subunit (GI164403; SEQ ID NO:7), produced using the multisequence alignment programof LASERGENE software (DNASTAR, Madison Wis.).

DESCRIPTION OF THE INVENTION

[0032] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0033] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0034] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, vectors, and methodologies which are reported in thepublications and which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

[0035] Definitions

[0036] “HPRM” refers to the amino acid sequences of purified HPRMobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and preferably the humanspecies, from any source, whether natural, synthetic, semi-synthetic, orrecombinant.

[0037] The term “agonist” refers to a molecule which, when bound toHPRM, increases or prolongs the duration of the effect of HPRM. Agonistsmay include proteins, nucleic acids, carbohydrates, or any othermolecules which bind to and modulate the effect of HPRM.

[0038] “Altered” nucleic acid sequences encoding HPRM include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polynucleotide the same HPRM or apolypeptide with at least one functional characteristic of HPRM.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HPRM, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HPRM. The encoded protein may also be“altered” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HPRM. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of BPRM isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

[0039] The terms “amino acid” or “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules. Inthis context, “fragments”, “immunogenic fragments”, or “antigenicfragments” refer to fragments of HPRM which are preferably about 5 toabout 15 amino acids in length and which retain some biological activityor immunological activity of HPRM. Where “amino acid sequence” isrecited herein to refer to an amino acid sequence of a naturallyoccurring protein molecule, “amino acid sequence” and like terms are notmeant to limit the amino acid sequence to the complete native amino acidsequence associated with the recited protein molecule.

[0040] “Amplification” relates to the production of additional copies ofa nucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach and Dveksler (1995) PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview N.Y., pp. 1-5).

[0041] The term “antagonist” refers to a molecule which, when bound toHPRM, decreases the amount or the duration of the effect of thebiological or immunological activity of HPRM. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HPRM.

[0042] The term “antibody” refers to intact molecules as well as tofragments thereof, such as Fa, F(ab′)₂, and Fv fragments, which arecapable of binding the epitopic determinant. Antibodies that bind HPRMpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0043] The term “antigenic determinant” refers to that fragment of amolecule, an epitope, that makes contact with a particular antibody.When a protein or a fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to antigenic determinants, givenregions or three-dimensional structures on the protein. An antigenicdeterminant may compete with the intact antigen, the immunogen used toelicit the immune response, for binding to an antibody.

[0044] The term “biologically active” refers to a protein havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic HPRM, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0045] The terms “complementary” or “complementarity” refer to thenatural binding of polynucleotides under permissive salt and temperatureconditions by base pairing. For example, the sequence “A-G-T” binds tothe complementary sequence “T-C-A”. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of the hybridization. This is of particular importance inamplification reactions, which depend upon binding between nucleic acidsstrands, and in the design and use of peptide nucleic acid (PNA)molecules. The inhibition of hybridization of a completely complementarysequence to the target sequence may be examined using a hybridizationassay (Southern or northern blot, solution hybridization, and the like)under conditions of reduced stringency.

[0046] A “composition comprising a given polynucleotide” or a“composition comprising a given polypeptide” refer broadly to anycomposition containing the given polynucleotide or polypeptide and atleast one other molecule. The other molecule specifically encompasseslabeling moeities, reporter molecules, and pharmaceutical excipients andcarriers.

[0047] “Consensus sequence” refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, extended using XL-PCR kit(Applied Biosystems (ABI), Foster City Calif.) in the 5′ and/or the 3′direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW Fragment Assemblysystem (Genetics Computer Group, Madison Wis.). Some sequences have beenboth extended and assembled to produce the consensus sequence.

[0048] The term “correlates with expression of a polynucleotide”indicates that the detection of the presence of nucleic acids, the sameor related to a polynucleotide encoding HPRM, by northern analysis isindicative of the presence of nucleic acids encoding HPRM in a sample,and thereby correlates with expression of the transcript from thepolynucleotide encoding HPRM.

[0049] A “deletion” refers to a change in the amino acid or nucleotidesequence that results in the absence of one or more amino acid residuesor nucleotides.

[0050] The term “derivative” refers to the chemical modification ofHPRM, of a polynucleotide encoding HPRM, or of a polynucleotidecomplementary to a polynucleotide encoding HPRM. Chemical modificationsof a polynucleotide can include, for example, replacement of hydrogen byan alkyl, acyl, or amino group. A derivative polynucleotide encodes apolypeptide which retains at least one biological or immunologicalfunction of the natural polypeptide. A derivative polypeptide is onemodified by glycosylation, pegylation, or any similar process thatretains at least one biological or immunological function of the naturalpolypeptide.

[0051] The term “homology” refers to a degree of similarity or“identity”. “Percent identity” refers to the percentage of sequenceidentity found in a comparison of two or more amino acid or nucleic acidsequences. Percent identity can be determined electronically, e.g., byusing the multi-sequence alignment program of LASERGENE software(DNASTAR). This program can create alignments between two or moresequences according to selected method, e.g., the clustal method(Higgins and Sharp (1988) Gene 73:237-244). The clustal algorithm groupssequences into clusters by examining the distances between all pairs.The clusters are aligned pairwise, and then, in groups. The percentageidentity between two amino acid sequences, e.g., sequence A and sequenceB, is calculated by dividing the length of sequence A, minus the numberof gap residues in sequence A, minus the number of gap residues insequence B, into the sum of the residue matches between sequence A andsequence B, times one hundred. Gaps of low or of no homology between thetwo amino acid sequences are not included in determining percentageidentity. Percent identity between nucleic acid sequences can also becounted or calculated by methods known in the art, e.g., the Jotun Heinmethod (Hein (1990) Methods Enzymol 183:626-645).

[0052] “Hybridization” refers to any process by which a strand ofnucleic acid binds with a complementary strand through base pairing.

[0053] The term “hybridization complex” refers to a complex formedbetween two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution or formed between one nucleic acid sequencepresent in solution and another nucleic acid sequence immobilized on asubstrate.

[0054] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0055] The phrases “nucleic acid” or “nucleic acid sequence” refer to anoligonucleotide, nucleotide, polynucleotide, or any fragment thereof, toDNA or RNA of genomic or synthetic origin which may be single-strandedor double-stranded and may represent the sense or the antisense strand,to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.In this context, “fragments” refers to those nucleic acid sequenceswhich are greater than about 60 nucleotides in length, and mostpreferably are at least about 100 nucleotides, at least about 1000nucleotides, or at least about 10,000 nucleotides in length.

[0056] The terms “operably associated” or “operably linked” refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the transcription of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

[0057] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to 60 nucleotides, preferably about 15 to30 nucleotides, and most preferably about 20 to 25 nucleotides, whichcan be used in PCR amplification or in a hybridization assay ormicroarray. Oligonucleotide is equivalent to the terms “amplimer”,“primer”, and “oligomer”.

[0058] “Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAand RNA and stop transcript elongation; they may be pegylated to extendtheir lifespan in the cell (Nielsen et al. (1993) Anticancer Drug Des8:53-63).

[0059] The term “sample” is used in its broadest sense. A biologicalsample suspected of containing nucleic acids encoding HPRM, or fragmentsthereof, or HPRM itself, may comprise a bodily fluid; an extract fromcell media, a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

[0060] “Specific binding” refers to a specific interaction between anucleotide or protein and molecules with which it interacts. Thesemolecules include, but are not limited to, DNA molecules, RNA molecules,peptide nucleic acids, artificial chromosome constructions, peptides,proteins, agonists, antibodies, antagonists, immunoglobulins,inhibitors, drug compounds, peptides, and pharmaceutical agents. Theinteraction between the polynucleotide or polypeptide and the boundmolecule is dependent upon the presence of a particular structure of thepolynucleotide or protein recognized by the binding molecule. Forexample, if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

[0061] The term “stringent conditions” refers to conditions which permithybridization between polynucleotide sequences and the claimedpolynucleotide sequences. Suitably stringent conditions can be definedby, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

[0062] The term “purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment or cellculture media and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are associated.

[0063] “Specific binding” refers to a specific interaction between anucleotide or protein and molecules with which it interacts. Thesemolecules include, but are not limited to, DNA molecules, RNA molecules,peptide nucleic acids, artificial chromosome constructions, peptides,proteins, agonists, antibodies, antagonists, immunoglobulins,inhibitors, drug compounds, peptides, and pharmaceutical agents. Theinteraction between the polynucleotide or polypeptide and the boundmolecule is dependent upon the presence of a particular structure of thepolynucleotide or protein recognized by the binding molecule. Forexample, if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

[0064] “Substrate” refers to any solid support including, but notlimited to, membranes, filters, chips, slides, wafers, fibers, magneticor nonmagnetic beads, gels, capillaries or other tubing, plates,polymers, and microparticles with a variety of surface forms includingwells, trenches, pins, channels and pores to which cells or theirnucleic acids have been attached.

[0065] A “variant” of HPRM refers to a nucleotide or amino acid sequencethat is altered by one or more nucleotides or amino acids. The variantmay have “conservative” changes wherein the substituted moiety hassimilar structural or chemical properties (e.g., a purine is substitutedfor a purine or a leucine is replaced with an isoleucine). Analogousminor variations may also include at least one amino acid deletion orinsertion, or both. Guidance in determining which amino acid residuesmay be substituted, inserted, or deleted without abolishing biologicalor immunological activity may be found using computer programs wellknown in the art such as LASERGENE software (DNASTAR).

[0066] The Invention

[0067] The invention is based on the discovery of new human proteinasemolecules (HPRM), the polynucleotides encoding HPRM and the use of thesecompositions for the diagnosis and treatment of cancer and immunedisorders.

[0068] Nucleic acids encoding the HPRM-1 of the present invention werefirst identified in Incyte Clone 456855 from the keratinocyte cDNAlibrary (KERANOT01) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:4, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 456855 (KERANOT01) and 3363138 (PROSBPT02).

[0069] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 as shown in FIGS.2A-2E. HPRM-1 is 248 amino acids in length and contains potentialphosphorylation sites for casein kinase II at T68, S73, S129, and S237,and for protein kinase C at T123, T136, and S237, and for tyrosinekinase at Y146. HPRM-1 also contains a potential EF-hand calcium-bindingdomain between residues D132 and F144. As shown in FIGS. 4A-4B, HPRM-1has chemical and structural homology with the calcium-binding, calpain Ilight subunit from pig (GI 164403; SEQ ID NO:7). In particular, HPRM-1and the pig calpain subunit share 65% homology. The pig calpain subunitshares the EF-hand calcium-binding domain, and the potentialphosphorylation sites found at residues T68, T123, and Y146 in HPRM-1. Afragment of SEQ ID NO:4 from about nucleotide 145 to about nucleotide193 is useful for hybridization. Northern analysis shows the expressionof this sequence in skin, neonatal keratinocytes, and hyperplasticprostate cDNA libraries.

[0070] Nucleic acids encoding the HPRM-2 of the present invention werefirst identified in Incyte Clone 947429 from the atrium tissue cDNAlibrary (RATRNOT02) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:5, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 947429 (RATRNOT02), 870803 and 877928 (LUNGAST01), 907964(COLNNOTO9), and 2632243 (COLNTUT15).

[0071] In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, as shown in FIGS.2A-2E. HPRM-2 is 415 amino acids in length and has a potential signalpeptide sequence between residues M1 and Q23. A potentialN-glycosylation site is found at residue N355, and potentialphosphorylation sites are found for casein kinase II at T64, S142, andT274, for protein kinase C at T60, T109, S164, S241, and S357, and fortyrosine kinase at Y207. Cysteine residues, representing potentialintramolecular disulfide bridging sites are found at residues C34, C59,C86, C107, C154, C181, C208, C231, C297, C312, C364, and C415. HPRM-2has chemical and structural homology with mouse procollagen C-proteinaseenhancer (GI 2589009;SEQ ID NO:8). In particular, HPRM-2 and mouseprocollagen C-proteinase enhancer share 42% homology, thephosphorylation sites at T64, T109, and S357, and the twelve cysteineresidues found in HPRM-2. A fragment of SEQ ID NO:5 from aboutnucleotide 405 to about nucleotide 513 is useful for hybridization.Northern analysis shows the expression of this sequence in variouslibraries, at least 45% of which are immortalized or cancerous and atleast 31% of which involve immune response. Of particular note is theexpression of HPRM in tumors of the testes, lung, heart, colon, andbladder, and in inflammatory conditions including rheumatoid arthritis,asthma, and Crohn's disease.

[0072] Nucleic acids encoding the HPRM-3 of the present invention werefirst identified in Incyte Clone 1516165 from the pancreatic tumor cDNAlibrary (PANCTUT01) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:6, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 1516165 (PANCTUT01), 1360069 (LUNGNOT12), 794210 (OVARNOT03), andshotgun sequences SAWA02729, SAWA00677, SAWA01399, and SAWA00459.

[0073] In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS.3A-3E. HPRM-3 is 349 amino acids in length and has a potential signalpeptide sequence from residue M1 to A17, a potential N-glycosylationsite at residue N90, and potential phosphorylation sites for caseinkinase II at S65, S168, T175, S221, T293, and S333, and for proteinkinase C at S31 and S65. HPRM-3 also contains a potential eukaryoticaspartyl protease active site signature sequence between residues V93and V104, in which D96 is the catalytic site. HPRM-3 has chemical andstructural homology with human cathepsin E precursor (GI 181194; SEQ IDNO:9). In particular, HPRM-3 and the human cathepsin E precursor share88% homology. HPRM-3 is an apparent splice variant of the humancathepsin E precursor in which the sequence of the latter moleculebetween residues 1263 and E309 has been deleted. The fragment of SEQ IDNO:6 from about nucleotide 807 to about nucleotide 857, whichencompasses this deletion, is useful for hybridization. Northernanalysis shows the expression of this sequence in various libraries, atleast 61% of which are immortalized or cancerous and at least 32% ofwhich involve immune response. Of particular note is the expression ofHPRM-3 in tumors of the ovaries, pancreas, testes, and lung, and ininflammatory conditions including asthma, lymphocytic thyroiditis, andinflamed adenoids.

[0074] The invention also encompasses HPRM variants. A preferred HPRMvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the BPRM amino acid sequence, and which contains at leastone functional or structural characteristic of HPRM.

[0075] The invention also encompasses polynucleotides which encode HPRM.In a particular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:4, as shown in FIGS.1A-1C, which encodes an HPRM. In a further embodiment, the inventionencompasses the polynucleotide sequence comprising the sequence of SEQID NO:5, as shown in FIGS. 2A-2E. In a further embodiment, the inventionencompasses the polynucleotide sequence comprising the sequence of SEQID NO:6, as shown in FIGS. 3A-3E.

[0076] The invention also encompasses a variant of a polynucleotidesequence encoding HPRM. In particular, such a variant polynucleotidesequence will have at least about 80%, more preferably at least about90%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding HPRM. A particularaspect of the invention encompasses a variant of SEQ ID NO:4 which hasat least about 80%, more preferably at least about 90%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:4. The invention further encompasses a polynucleotide variant of SEQID NO:5 having at least about 80%, more preferably at least about 90%,and most preferably at least about 95% polynucleotide sequence identityto SEQ ID NO:5. The invention further encompasses a polynucleotidevariant of SEQ ID NO:6 having at least about 80%, more preferably atleast about 90%, and most preferably at least about 95% polynucleotidesequence identity to SEQ ID NO:6. Any one of the polynucleotide variantsdescribed above can encode an amino acid sequence which contains atleast one functional or structural characteristic of HPRM.

[0077] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding HPRM, some bearing minimal homology tothe polynucleotide sequences of any known and naturally occurring gene,may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringHPRM, and all such variations are to be considered as being specificallydisclosed.

[0078] Although nucleotide sequences which encode HPRM and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HPRM under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HPRM or its derivatives possessing a different codon usage.Codons may be selected to increase the rate at which expression of thepeptide occurs in a particular prokaryotic or eukaryotic host inaccordance with the frequency with which particular codons are utilizedby the host. Other reasons for altering the nucleotide sequence encodingHPRM and its derivatives without altering the encoded amino acidsequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0079] The invention also encompasses production of DNA sequences whichencode HPRM and HPRM derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding HPRM or any fragment thereof.

[0080] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NOs:4-6 or fragmentsthereof under various conditions of stringency (Wahl and Berger (1987)Methods Enzymol 152:399-407; Kimmel (1987) Methods Enzymol 152:507-511).

[0081] Methods for DNA sequencing are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE, Taq polymerase, thermostable T7 polymerase(Amersham Pharmacia Biotech (APB), Piscataway N.J.), or combinations ofpolymerases and proofreading exonucleases such as those found in theELONGASE amplification system (Life Technologies, Rockville Md.).Preferably, the process is automated with machines such as the MICROLAB2200 (Hamilton, Reno Nev.), DNA ENGINE thermal cycler (MJ Research,Waltham Mass.) and the CATALYST and 373 and 377 PRISM DNA sequencingsystems (ABI).

[0082] The nucleic acid sequences encoding HPRM may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar (1993) PCR Methods Applic2:318-322). In particular, genomic DNA is first amplified in thepresence of a primer which is complementary to a linker sequence withinthe vector and a primer specific to a region of the nucleotide sequence.The amplified sequences are then subjected to a second round of PCR withthe same linker primer and another specific primer internal to the firstone. Products of each round of PCR are transcribed with an appropriateRNA polymerase and sequenced using reverse transcriptase.

[0083] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al. (1988) NucleicAcids Res 16:8186). The primers may be designed using commerciallyavailable software such as OLIGO software (Molecular Biology Insights,Cascade Colo.) or another appropriate program to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to 72° C.The method uses several restriction enzymes to generate a fragment inthe known region of a gene. The fragment is then circularized byintramolecular ligation and used as a PCR template.

[0084] Another method which may be used is capture PCR, which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom et al (1991) PCR MethodsApplic 1:111-119). In this method, multiple restriction enzymedigestions and ligations may be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art (Parker et al. (1991) NucleicAcids Res 19:3055-3060). Additionally, one may use PCR, nested primers,and PROMOTERF NDER libraries to walk genomic DNA (Clontech, Palo AltoCalif.). This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

[0085] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0086] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and a charge coupled device camera for detection of theemitted wavelengths. Output/light intensity may be converted toelectrical signal using appropriate software (e.g., GENOTYPER andSEQUENCE NAVIGATOR software, ABI), and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0087] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode HPRM may be used in recombinant DNAmolecules to direct expression of HPRM, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encode thesame or a functionally equivalent amino acid sequence may be produced,and these sequences may be used to clone and express HPRM.

[0088] As will be understood by those of skill in the art, it may beadvantageous to produce HPRM-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0089] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterHPRM-encoding sequences for a variety of reasons including, but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0090] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HPRM may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HPRM activity, it may be useful toencode a chimeric HPRM protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HPRM encoding sequence and theheterologous protein sequence, so that HPRM may be cleaved and purifiedaway from the heterologous moiety.

[0091] In another embodiment, sequences encoding HPRM may besynthesized, in whole or in part, using chemical methods well known inthe art (Caruthers et al (1980) Nucleic Acids Symp Ser (7) 215-223; Hornet al. (1980) Nucleic Acids Symp Ser (7) 225-232). Alternatively, theprotein itself maybe produced using chemical methods to synthesize theamino acid sequence of HPRM, or a fragment thereof. For example, peptidesynthesis can be performed using various solid-phase techniques (Robergeet al. (1995) Science 269:202-204). Automated synthesis may be achievedusing the 431A peptide synthesizer (ABI). Additionally, the amino acidsequence of HPRM, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide.

[0092] The peptide may be purified by preparative high performanceliquid chromatography (Chiez and Regnier (1990) Methods Enzymol182:392-421). The composition of the synthetic peptides may be confirmedby amino acid analysis or by sequencing (Creighton (1984) Proteins,Structures and Molecular Properties, W H Freeman, New York N.Y.).

[0093] In order to express a biologically active HPRM, the nucleotidesequences encoding HPRM or derivatives thereof may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0094] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding HPRMand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel et al. (1995, andperiodic supplements) Current Protocols in Molecular Biology, John Wiley& Sons, New York N.Y., ch. 9, 13, and 16.)

[0095] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding HPRM. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0096] The “control elements” or “regulatory sequences” are thosenon-translated regions, e.g., enhancers, promoters, and 5′ and 3′untranslated regions, of the vector and polynucleotide sequencesencoding HPRM which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of transcription and translation elements, including constitutiveand inducible promoters, may be used. For example, when cloning inbacterial systems, inducible promoters, e.g., hybrid lacZ promoter ofthe BLUESCRIPT phagemid (Stratagene, La Jolla Calif.) or PSPORT1 plasmid(Life Technologies), may be used. The baculovirus polyhedrin promotermay be used in insect cells. Promoters or enhancers derived from thegenomes of plant cells (e.g., heat shock, RUBISCO, and storage proteingenes) or from plant viruses (e.g., viral promoters or leader sequences)may be cloned into the vector. In mammalian cell systems, promoters frommammalian genes or from mammalian viruses are preferable. If it isnecessary to generate a cell line that contains multiple copies of thesequence encoding HPRM, vectors based on SV40 or EBV may be used with anappropriate selectable marker.

[0097] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for HPRM. For example, whenlarge quantities of HPRM are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, multifunctional E. coli cloning and expression vectors such asBLUESCRIPT phagemid (Stratagene), in which the sequence encoding HPRMmay be ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced, and pin vectors. (Van Heeke andSchuster (1989) J Biol Chem 264:5503-5509). PGEX vectors (APB) may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0098] In the yeast Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters, such as alpha factor,alcohol oxidase, and PGH, may be used (Ausubel, supra; Grant et al.(1987) Methods Enzymol 153:516-544).

[0099] In cases where plant expression vectors are used, the expressionof sequences encoding HPRM may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu (1987) EMBO J 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al. (1984) EMBO J3:1671-1680; Broglie et al. (1984) Science 224:838-843; and Winter etal. (1991) Results Probl Cell Differ 17:85-105). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (Hobbs or Murry (1992) In:Yearbook of Science and Technology, McGraw Hill, New York N.Y.; pp.191-196).

[0100] An insect system may also be used to express HPRM. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding HPRMmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of sequences encoding HPRM will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses may then be used to infect, forexample, S. frugiperda cells or Trichoplusia larvae in which HPRM maybeexpressed (Engelhard et al. (1994) Proc Nat Acad Sci 91:3224-3227).

[0101] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding HPRM may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing HPRM in infected host cells (Logan andShenk (1984) Proc Natl Acad Sci 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0102] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

[0103] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding HPRM. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding HPRM and its initiation codon and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used (Scharf et al. (1994) Results Probl Cell Differ20:125-162).

[0104] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are available fromthe ATCC (Bethesda Md.) and may be chosen to ensure the correctmodification and processing of the foreign protein.

[0105] For long term, high yield production of recombinant proteins,stable expression is preferred. For example, cell lines capable ofstably expressing HPRM can be transformed using expression vectors whichmay contain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0106] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase genes and adeninephosphoribosyltransferase genes, which can be employed in tk or aprcells, respectively (Wigler et al. (1977) Cell 11:223-232; Lowy et al.(1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or herbicideresistance can be used as the basis for selection. For example, dhfrconfers resistance to methotrexate; npt confers resistance to theaminoglycosides neomycin and G-418; and als or pat confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Wigler et al. (1980) Proc Natl Acad Sci 77:3567-3570; Colbere-Garapinet al. (1981) J Mol Biol 150:1-14; and Murry, supra). Additionalselectable genes have been described, e.g., trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman and Mulligan (1988) ProcNatl Acad Sci 85:8047-8051). Visible markers such as anthocyanins, βglucuronidase and its substrate GUS, luciferase and its substrateluciferin may be used. Green fluorescent proteins (GFP; Clontech) canalso be used. These markers can be used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes etal. (1995) Methods Mol Biol 55:121-131).

[0107] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding HPRM is inserted within a marker gene sequence, transformedcells containing sequences encoding HPRM can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding HPRM under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0108] Alternatively, host cells which contain the nucleic acid sequenceencoding HPRM and express HPRM may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

[0109] The presence of polynucleotide sequences encoding HPRM can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding HPRM.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding HPRM todetect transformants containing DNA or RNA encoding HPRM.

[0110] A variety of protocols for detecting and measuring the expressionof HPRM, using either polyclonal or monoclonal antibodies specific forthe protein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HPRM is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art (Hampton et al. (1990) Serological Methods, a LaboratoryManual, APS Press, St Paul Minn., Section IV; Maddox et al. (1983) J ExpMed 158:1211-1216).

[0111] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding HPRMinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding HPRM, or any fragments thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits such as those provided by APB.Reporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0112] Host cells transformed with nucleotide sequences encoding HPRMmay be cultured under conditions for the expression and recovery of theprotein from cell culture. The protein produced by a transformed cellmay be secreted or contained intracellularly depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing polynucleotides which encode HPRM maybe designed to contain signal sequences which direct secretion of HPRMthrough a prokaryotic or eukaryotic cell membrane. Other constructionsmay be used to join sequences encoding HPRM to nucleotide sequencesencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA (APB)or enterokinase (Invitrogen, Carlsbad Calif.), between the purificationdomain and the HPRM encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing HPRM and a nucleic acid encoding 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on immobilized metal ionaffinity chromatography (Porath et al. (1992) Prot Exp Purif 3:263-281). The enterokinase cleavage site provides a means for purifyingHPRM from the fusion protein (Kroll et al. (1993) DNA Cell Biol12:441-453).

[0113] Fragments of HPRM may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques (Creighton (1984) Protein: Structures and MolecularProperties, pp. 55-60, W H Freeman, New York N.Y.). Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using the 431A peptidesynthesizer (ABI). Various fragments of HPRM may be synthesizedseparately and then combined to produce the full length molecule.

[0114] Therapeutics

[0115] Chemical and structural homology exists among HPRM and thecalcium-binding, calpain I subunit from pig (GI 164403), a procollagen-Cproteinase enhancer protein from mouse (GI 2589009), and an asparticproteinase, cathepsin E, from human (GI 181194). In addition, HPRM isexpressed in cancer and the immune response. Therefore, HPRM appears toplay a role in cancer and immune disorders.

[0116] Therefore, in one embodiment, an antagonist of HPRM may beadministered to a subject to treat or prevent a cancer. Such a cancermay include, but is not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds HPRM may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express HPRM.

[0117] In another embodiment, a vector expressing the complement of thepolynucleotide encoding HPRM may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

[0118] In another embodiment, an antagonist of HPRM may be administeredto a subject to treat or prevent an immune disorder. Such an immunedisorder may include, but is not limited to, AIDS, Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome,systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,ulcerative colitis, Werner syndrome, and complications of cancer,hemodialysis, and extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections; and trauma.

[0119] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding HPRM may be administered to a subject totreat or prevent an immune disorder including, but not limited to, thosedescribed above.

[0120] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

[0121] An antagonist of HPRM may be produced using methods which aregenerally known in the art. In particular, purified HPRM may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind HPRM. Antibodies to HPRM may alsobe generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, thosewhich inhibit dimer formation, are especially preferred for therapeuticuse.

[0122] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HPRM or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0123] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to HPRM have an amino acid sequence consistingof at least about 5 amino acids, and, more preferably, of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofHPRM amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0124] Monoclonal antibodies to HPRM may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler et al. (1975) Nature 256:495-497; Kozboret al. (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc NatlAcad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120).

[0125] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison et al. (1984) Proc NatlAcad Sci 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; andTakeda et al. (1985) Nature 314:452-454). Alternatively, techniquesdescribed for the production of single chain antibodies may be adapted,using methods known in the art, to produce HPRM-specific single chainantibodies. Antibodies with related specificity, but of distinctidiotypic composition, may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci88:10134-10137).

[0126] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi et al. (1989) Proc Natl Acad Sci 86: 3833-3837;Winter et al. (1991) Nature 349:293-299).

[0127] Antibody fragments which contain specific binding sites for HPRMmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (Huse et al.(1989) Science 246:1275-1281).

[0128] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between HPRM and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HPRM epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra.).

[0129] In another embodiment of the invention, the polynucleotidesencoding HPRM, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding HPRM may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding HPRM. Thus, complementary molecules or fragments may be used tomodulate HPRM activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments can be designed from variouslocations along the coding or control regions of sequences encodingHPRM.

[0130] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors which will express nucleic acidsequences complementary to the polynucleotides of the gene encoding HPRM(Sambrook, supra; Ausubel, supra).

[0131] Genes encoding HPRM can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding HPRM. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0132] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5, or regulatory regions of the geneencoding HPRM. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee et al. (1994) In: Huber and Carr,Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y.,pp. 163-177). A complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0133] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHPRM.

[0134] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0135] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding HPRM. Such DNA sequences may be incorporated into a widevariety of vectors with RNA polymerase promoters such as T7 or SP6.Alternatively, these cDNA constructs that synthesize complementary RNA,constitutively or inducibly, can be introduced into cell lines, cells,or tissues.

[0136] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5 ′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thynine, anduridine which are not as easily recognized by endogenous endonucleases.

[0137] Many methods for introducing vectors into cells or tissues areavailable for use in vivo, in vitro, and ex vivo. For ex vivo therapy,vectors may be introduced into stem cells taken from the patient andclonally propagated for autologous transplant back into that samepatient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art (Goldman et al. (1997) Nature Biotechnol 15:462-466).

[0138] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0139] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of HPRM, antibodies to HPRM, and mimetics, agonists,antagonists, or inhibitors of HPRM. The compositions may be administeredalone or in combination with at least one other agent, such as astabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0140] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0141] In addition to the active ingredients, these pharmaceuticalcompositions may contain pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Reminton's Pharmaceutical Sciences (MaackPublishing, Easton Pa.).

[0142] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

[0143] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Excipients include carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, andsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; gums, including arabic and tragacanth;and proteins, such as gelatin and collagen. If desired, disintegratingor solubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate. Auxiliaries can be added, if desired.

[0144] Dragee cores may be used in conjunction with coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or dragee coatings forproduct identification or to characterize the quantity of activecompound, i.e., dosage.

[0145] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

[0146] Pharmaceutical formulations for parenteral administration may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Lipophilic solvents or vehicles include fattyoils, such as sesame oil, or synthetic fatty acid esters, such as ethyloleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain stabilizers or agents to increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions.

[0147] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0148] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0149] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0150] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of HPRM, such labeling wouldinclude amount, frequency, and method of administration.

[0151] Pharmaceutical compositions for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0152] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0153] A therapeutically effective dose refers to that amount of activeingredient, for example HPRM or fragments thereof, antibodies of HPRM,and agonists, antagonists or inhibitors of HPRM, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index. Pharmaceuticalcompositions which exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies are used toformulate a range of dosage for human use. The dosage contained in suchcompositions is preferably within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, thesensitivity of the patient, and the route of administration.

[0154] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0155] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0156] Diagnostics

[0157] In another embodiment, antibodies which specifically bind HPRMmay be used for the diagnosis of disorders characterized by expressionof HPRM, or in assays to monitor patients being treated with HPRM oragonists, antagonists, or inhibitors of HPRM. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for BPRM include methods whichutilize the antibody and a label to detect HPRM in human body fluids orin extracts of cells or tissues. The antibodies may be used with orwithout modification, and may be labeled by covalent or non-covalentattachment of a reporter molecule. A wide variety of reporter molecules,several of which are described above, are known in the art and may beused.

[0158] A variety of protocols for measuring HPRM, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of HPRM expression. Normal or standard valuesfor HPRM expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to HPRM under conditions for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of HPRM expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

[0159] In another embodiment of the invention, the polynucleotidesencoding HPRM may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof HPRM may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of HPRM, and tomonitor regulation of HPRM levels during therapeutic intervention.

[0160] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HPRM or closely related molecules may be used to identifynucleic acid sequences which encode HPRM. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HPRM, alleles, orrelated sequences.

[0161] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of theHPRM encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequences of SEQID NOs:4-6 or from genomic sequences including promoters, enhancers, andintrons of the HPRM gene.

[0162] Means for producing specific hybridization probes for DNAsencoding HPRM include the cloning of polynucleotide sequences encodingHPRM or HPRM derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

[0163] Polynucleotide sequences encoding HPRM may be used for thediagnosis of a disorder associated with expression of HPRM. Examples ofsuch a disorder include, but are not limited to, cancer, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immune disorders suchas AIDS, Addison's disease, adult respiratory distress syndrome,allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis,bronchitis, cholecystitis, contact dermatitis, Crohn'sdisease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, myocardial or pericardialinflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis,rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemicanaphylaxis, systemic lupus erythematosus, systemic sclerosis,ulcerative colitis, Werner syndrome, and complications of cancer,hemodialysis, and extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections; and trauma. Thepolynucleotide sequences encoding HPRM may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patients to detect altered HPRMexpression. Such qualitative or quantitative methods are well known inthe art.

[0164] In a particular aspect, the nucleotide sequences encoding HPRMmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding HPRM may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions for the formation ofhybridization complexes. After an incubation period, the sample iswashed and the signal is quantitated and compared with a standard value.If the amount of signal in the patient sample is significantly alteredin comparison to a control sample then the presence of altered levels ofnucleotide sequences encoding HPRM in the sample indicates the presenceof the associated disorder. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or to monitor the treatment of anindividual patient.

[0165] In order to provide a basis for the diagnosis of a disorderassociated with expression of HPRM, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding HPRM, underconditions for hybridization or amplification. Standard hybridizationmay be quantified by comparing the values obtained from normal subjectswith values from an experiment in which a known amount of a purifiedpolynucleotide is used. Standard values obtained in this manner may becompared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

[0166] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0167] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0168] Additional diagnostic uses for oligonucleotides designed from thesequences encoding HPRM may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HPRM, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HPRM, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0169] Methods which may also be used to quantitate the expression ofHPRM include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and interpolating results from standardcurves (Melby et al. (1993) J Immunol Methods 159:235-244; Duplaa et al.(1993) Anal Biochem 229-236). The speed of quantitation of multiplesamples may be accelerated by running the assay in an ELISA format wherethe oligomer of interest is presented in various dilutions and aspectrophotometric or colorimetric response gives rapid quantitation.

[0170] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0171] Microarrays may be prepared, used, and analyzed using methodsknown in the art (Brennan, et al. (1995) U.S. Pat. No. 5,474,796; Schenaet al. (1996) Proc Natl Acad Sci 93:10614-10619; Baldeschweiler et al.(1995) PCT application WO95/251116; Shalon et al. (1995) PCT applicationWO95/35505; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; andHeller et al. (1997) U.S. Pat. No. 5,605,662).

[0172] In another embodiment of the invention, nucleic acid sequencesencoding HPRM may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries (Price (1993) Blood Rev 7:127-134; Trask(1991) Trends Genet 7:149-154).

[0173] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data(Heinz-Ulrich et aL. (1995) In: Meyers, Molecular Biology andBiotechnology, VCH Publishers New York N.Y., pp. 965-968). Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HPRM on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

[0174] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., AT to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation (Gatti et al. (1988) Nature 336:577-580). The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc., amongnormal, carrier, or affected individuals.

[0175] In another embodiment of the invention, HPRM, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between HPRMand the agent being tested may be measured.

[0176] Another technique for drug screening provides for high throughputscreening of compounds having binding affinity to the protein ofinterest (Geysen, et al. (1984) PCT application WO84/03564). In thismethod, large numbers of different small test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thetest compounds are reacted with HPRM, or fragments thereof, and washed.Bound HPRM is then detected by methods well known in the art. PurifiedHPRM can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0177] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding HPRMspecifically compete with a test compound for binding HPRM. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with HPRM.

[0178] In additional embodiments, the nucleotide sequences which encodeHPRM may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0179] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0180] I RATRNOT02 cDNA Library Construction

[0181] The right atrium tissue used for the RATRNOT02 libraryconstruction was obtained from a 39 year old Caucasian male who died ofa gun shot wound. The frozen tissue was homogenized and lysed using aPOLYTRON homogenizer (Brinkmann Instruments, Westbury N.Y.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCI cushion using an SW28 rotor in a L8-70M ultracentrifuge(Beckman Coulter, Fullerton Calif.) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with phenol chloroform, pH4.0, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water and treated with DNase at 37° C. RNAextraction and precipitation were repeated as before. The MRNA wasisolated with the OLIGOTEX kit (Qiagen, Valencia Calif.) and used toconstruct the cDNA library.

[0182] A 10 million clone cDNA library was constructed using threemicrograms of poly A⁺ mRNA and Not I/oligo d(T) primer. The cDNAs weredirectionally inserted into Sal I/Not I sites of PSPORT1 plasmid (LifeTechnologies) and the plasmid was transformed into competent DH5α cellsor ELECTROMAX DH10B cells (Life Technologies).

[0183] II Isolation and Sequencing of cDNA Clones

[0184] Plasmid DNA was released from the cells and purified using theMINIPREP kit (Advanced Genetic Technologies, Gaithersburg Md.). This kitconsists of a 96-well block with reagents for 960 purifications. Therecommended protocol was employed except for the following changes: 1)the 96 wells were each filled with 1 ml of sterile TERRIHC BROTH (BDBiosciences, Sparks Md.) containing carbenicillin at 25 mg/l andglycerol at 0.4%; 2) the bacteria were inoculated into the wells,cultured for 24 hours, and then lysed with 60 μl of lysis buffer; 3) theblocks were centrifuged in the GS-6R rotor (Beckman Coulter) at 2900 rpmfor 5 minutes, and the contents of the block were added to the primaryfilter plate; and 4) the optional step of adding isopropanol to TRISbuffer was eliminated. After the last step in the protocol, samples weretransferred to a 96-well block for storage.

[0185] The cDNAs were prepared using a MICROLAB 2200 (Hamilton) incombination with DNA ENGINE thermal cyclers (MJ Research) and sequencedby the method of Sanger and Coulson (1975; J Mol Biol 94:441f) using 377PRISM DNA sequencing systems (ABI); and the reading frame wasdetermined.

[0186] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0187] The nucleotide sequences and/or amino acid sequences of theSequence Listing were used to query sequences in the GenBank, SwissProt,BLOCKS, and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool; Altschul (1993)J Mol Evol 36:290-300; Altschul et al. (1990) J Mol Biol 215:403-410).

[0188] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms could have been used when dealing with primary sequencepatterns and secondary structure gap penalties (Smith et al. (1992)Protein Engineering 5:35-51). The sequences disclosed in thisapplication have lengths of at least 49 nucleotides and have no morethan 12% uncalled bases (where N is rpalces A, C, G, or T).

[0189] The BLAST approach searched for matches between a query sequenceand a database sequence. BLAST evaluated the statistical significance ofany matches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

[0190] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam), and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

[0191] Additionally, sequences identified from cDNA libraries may beanalyzed to identify those gene sequences encoding conserved proteinmotifs using an appropriate analysis program, e.g., the Block 2Bioanalysis program (Incyte Genomics, Palo Alto Calif.). This motifanalysis program, based on sequence information contained in theSwissProt Database and PROSITE, is a method of determining the functionof uncharacterized proteins translated from genomic or cDNA sequences(Bairoch et al. (1997) Nucleic Acids Res 25:217-221; Attwood et al.(1997) J Chem Inf Comput Sci 37:417-424). PROSITE maybe used to identifycommon functional or structural domains in divergent proteins. Themethod is based on weight matrices. Motifs identified by this method arethen calibrated against the SwissProt database in order to obtain ameasure of the chance distribution of the matches.

[0192] In another alternative, Hidden Markov models (HMMs) maybe used tofind protein domains, each defined by a data set of proteins known tohave a common biological function (Pearson and Lipman (1988) Proc NatlAcad Sci 85:2444-2448; Smith and Waterman (1981) J Mol Biol147:195-197). HMMs were initially developed to examine speechrecognition patterns, but are now being used in a biological context toanalyze protein and nucleic acid sequences as well as to model proteinstructure (Keogh et al. (1994) J Mol Biol 235:1501-1531; Collin et al.(1993) Protein Sci 2:305-314). HMMs have a formal probabilistic basisand use position-specific scores for amino acids or nucleotides. Thealgorithm continues to incorporate information from newly identifiedsequences to increase its motif analysis capabilities.

[0193] IV Northern Analysis

[0194] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook, supra, ch. 7;Ausubel, supra, ch. 4 and 16).

[0195] Analogous computer techniques applying BLAST are used to searchfor identical or related molecules in nucleotide databases such asGenBank or LIFESEQ database (Incyte Genomics). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or homologous.

[0196] The basis of the search is the product score, which is definedas:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0197] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Homologous molecules are usually identified by selecting thosewhich show product scores between 15 and 40, although lower scores mayidentify related molecules.

[0198] The results of northern analysis are reported as a list oflibraries in which the transcript encoding HPRM occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0199] V Extension of HPRM Encoding Polynucleotides

[0200] The nucleic acid sequences of Incyte Clones 456855, 947429, and1516165 were used to design oligonucleotide primers for extendingpartial nucleotide sequences to full length. For each nucleic acidsequence, one primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence “outward” generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO software(Molecular Biology Insights), or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the target sequence at temperatures of about68° C. to about 72° C. Any stretch of nucleotides which would result inhairpin structures and primer-primer dimerizations was avoided.

[0201] Selected human cDNA libraries (Life Technologies) were used toextend the sequence. If more than one extension is necessary or desired,additional sets of primers are designed to further extend the knownregion.

[0202] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (ABI) and thoroughly mixing the enzymeand reaction mix. PCR was performed using the DNA ENGINE thermal cycler(MJ Research), beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, with the followingparameters: Step 1, 94° C. for 1 min (initial denaturation); Step 2, 65°C. for 1 min; Step 3, 68° C. for 6 min; Step 4, 94° C. for 15 sec; Step5, 65° C. for 1 min; Step 6, 68° C. for 7 min; Step 7, repeat steps 4through 6 for an additional 15 cycles; Step 8, 94° C. for 15 sec; Step9, 65° C. for 1 min; Step 10, 68° C. for 7:15 min; Step 11, repeat steps8 through 10 for an additional 12 cycles; Step 12, 72° C. for 8 min; andStep 13, 4° C. (and holding).

[0203] A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (Qiagen), and trimmed of overhangsusing Klenow enzyme to facilitate religation and cloning.

[0204] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium (Sambrook, supra,Appendix A, p. 2). After incubation for one hour at 37° C., the E. colimixture was plated on Luria Bertani (LB) agar (Sambrook, supra, AppendixA, p. 1) containing carbenicillin (2×carb). The following day, severalcolonies were randomly picked from each plate and cultured in 150 μl ofliquid LB/2×carb medium placed in an individual well of an appropriatecommercially-available sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture was transferred into a non-sterile96-well plate and, after dilution 1:10 with water, 5 μl from each samplewas transferred into a PCR array.

[0205] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1, 94° C. for 60 sec; Step 2, 94° C. for 20 sec; Step3, 55° C. for 30 sec; Step 4, 72° C. for 90 sec; Step 5, repeat steps 2through 4 for an additional 29 cycles; Step 6, 72° C. for 180 sec; andStep 7, 4° C. (and holding).

[0206] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0207] In like manner, the nucleotide sequences of SEQ ID NOs:4-6 areused to obtain 5′regulatory sequences using the procedure above,oligonucleotides designed for 5′extension, and an appropriate genomiclibrary.

[0208] VI Labeling and Use of Individual Hybridization Probes

[0209] Hybridization probes derived from SEQ ID NO:4-6 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO software (Molecular Biology Insights) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (APB), and T4 polynucleotide kinase (PerkinElmer LifeSciences, Boston Mass.). The labeled oligonucleotides are purified usinga SEPHADEX G-25 superfine resin column (APB). An aliquot containing 10⁷counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal,or Pvu II (PerkinElmer Life Sciences).

[0210] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to NYTRAN PLUS membranes (Schleicher & Schuell,Keene N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Eastman Kodak, Rochester N.Y.) is exposed to the blots for severalhours, hybridization patterns are compared.

[0211] VII Microarrays

[0212] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate(Baldeschweiler, supra). An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0213] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsthereof may comprise the elements of the microarray. Fragments forhybridization can be selected using software well known in the art suchas LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragmentsthereof corresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying (Schena et al. (1995) Science 270:467-470; Shalon et al. (1996)Genome Res 6:639-645). Fluorescent probes are prepared and used forhybridization to the elements on the substrate. The substrate isanalyzed by procedures described above.

[0214] VIII Complementary Polynucleotides

[0215] Sequences complementary to the HPRM-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HPRM. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO software(Molecular Biology Insights) and the coding sequence of HPRM. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the HPRM-encoding transcript.

[0216] IX Expression of HPRM

[0217] Expression of HPRM is accomplished by subcloning the cDNA into anappropriate vector and transforming the vector into host cells. Thisvector contains an appropriate promoter, e.g., B-galactosidase, upstreamof the cloning site, operably associated with the cDNA of interest(Sambrook, supra, pp. 404-433; Rosenberg et al (1983) Methods Enzymol101:123-138).

[0218] Induction of an isolated, transformed bacterial strain withisopropyl beta-D-thiogalactopyranoside (IPTG) using standard methodsproduces a fusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of HPRM into bacterialgrowth media which can be used directly in the following assay foractivity.

[0219] X Demonstration of HPRM Activity

[0220] Protease activity of HPRM is measured by the hydrolysis ofappropriate synthetic peptide substrates conjugated with variouschromogenic molecules in which the degree of hydrolysis is quantitatedby spectrophotometric (or fluorometric) absorption of the releasedchromophore (Beynon and Bond, supra pp.25-55). Peptide substrates aredesigned according to the category of protease activity as endopeptidase(serine, cysteine, aspartic proteases), animopeptidase (leucineaminopeptidase), or carboxypeptidase (carboxypeptidase A and B,procollagen C-proteinase). Chromogens commonly used are 2-naphthylamine,4-nitroaniline, and furylacrylic acid. Assays are performed at roomtemperature (˜25° C.) and contain an aliquot of the enzyme and theappropriate substrate in a buffer. Reactions are carried out in anoptical cuvette and followed by the increase/decrease in absorbance ofthe chromogen released during hydrolysis of the peptide substrate. Thechange in absorbance is proportional to the enzyme activity in theassay.

[0221] Enhancement of procollagen C-proteinase activity (HPRM-2) isdetermined by measuring procollagen C-proteinase activity in the absenceand presence of enhancer protein. Procollagen C-proteinase activity ismeasured as described above using an appropriate carboxypeptidasesubstrate in the absence and in the presence of varying amounts ofHPRM-2. The increase in activity of procollagen C-proteinase measured inthe presence of HPRM-2 compared to that measured in its absence isproportional to the activity of HPRM-2 in the assay.

[0222] XI Production of HPRM Specific Antibodies

[0223] HPRM is purified using PAGE electrophoresis (Harrington(1990)Methods Enzymol 182:488-495), or other purification techniques, is usedto immunize rabbits and to produce antibodies using standard protocols.

[0224] Alternatively, the HPRM amino acid sequence is analyzed usingLASERGENE software (DNASTAR) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art (Ausubel, supra,ch. 11).

[0225] Typically, oligopeptides 15 residues in length are synthesizedusing a Model 431A peptide synthesizer (ABI) using fmoc-chemistry andcoupled to KLH (Sigma-Aldrich, St.Louis Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity (Ausubel, supra.). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

[0226] XII Purification of Naturally Occurring HPRM Using SpecificAntibodies

[0227] Naturally occurring or recombinant HPRM is purified byimmunoaffinity chromatography using antibodies specific for HPRM. Animmunoaffinity column is constructed by covalently coupling anti-HPRMantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE resin (APB). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

[0228] Media containing HPRM are passed over the immunoaffinity column,and the column is washed under conditions, e.g., in high ionic strengthbuffers in the presence of detergent, that allow the preferentialabsorbance of HPRM. The column is eluted under conditions, e.g., abuffer of pH 2 to pH 3, or a high concentration of a chaotrope, such asurea or thiocyanate ion, that disrupt antibody/HPRM binding, and HPRM iscollected.

[0229] XIII Identification of Molecules Which Interact with HPRM

[0230] HPRM, or biologically active fragments thereof, are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem J 133:529-533).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HPRM, washed, and any wells withlabeled HPRM complex are assayed. Data obtained using differentconcentrations of HPRM are used to calculate values for the number,affinity, and association of HPRM with the candidate molecules.

[0231] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1 9 1 248 PRT Homo sapiens misc_feature Incyte ID No 456855 1 Met PheLeu Ala Lys Ala Leu Leu Glu Gly Ala Asp Arg Gly Leu 1 5 10 15 Gly GluAla Leu Gly Gly Leu Phe Gly Gly Gly Gly Gln Arg Arg 20 25 30 Glu Gly GlyGly Arg Asn Ile Gly Gly Ile Val Gly Gly Ile Val 35 40 45 Asn Phe Ile SerGlu Ala Ala Ala Ala Gln Tyr Thr Pro Glu Pro 50 55 60 Pro Pro Thr Gln GlnHis Phe Thr Ser Val Glu Ala Ser Glu Ser 65 70 75 Glu Glu Val Arg Arg PheArg Gln Gln Phe Thr Gln Leu Ala Gly 80 85 90 Pro Asp Met Glu Val Gly AlaThr Asp Leu Met Asn Ile Leu Asn 95 100 105 Lys Val Leu Ser Lys His LysAsp Leu Lys Thr Asp Gly Phe Ser 110 115 120 Leu Asp Thr Cys Arg Ser IleVal Ser Val Met Asp Ser Asp Thr 125 130 135 Thr Gly Lys Leu Gly Phe GluGlu Phe Lys Tyr Leu Trp Asn Asn 140 145 150 Ile Lys Lys Trp Gln Cys ValTyr Lys Gln Tyr Asp Arg Asp His 155 160 165 Ser Gly Ser Leu Gly Ser SerGln Leu Arg Gly Ala Leu Gln Ala 170 175 180 Ala Gly Phe Gln Leu Asn GluGln Leu Tyr Gln Met Ile Val Arg 185 190 195 Arg Tyr Ala Asn Glu Asp GlyAsp Met Asp Phe Asn Asn Phe Ile 200 205 210 Ser Cys Leu Val Arg Leu AspAla Met Phe Arg Ala Phe Lys Ser 215 220 225 Leu Asp Arg Asp Arg Asp GlyLeu Ile Gln Val Ser Ile Lys Glu 230 235 240 Trp Leu Gln Leu Thr Met TyrSer 245 2 415 PRT Homo sapiens misc_feature Incyte ID No 947429 2 MetArg Gly Ala Asn Ala Trp Ala Pro Leu Cys Leu Leu Leu Ala 1 5 10 15 AlaAla Thr Gln Leu Ser Arg Gln Gln Ser Pro Glu Arg Pro Val 20 25 30 Phe ThrCys Gly Gly Ile Leu Thr Gly Glu Ser Gly Phe Ile Gly 35 40 45 Ser Glu GlyPhe Pro Gly Val Tyr Pro Pro Asn Ser Lys Cys Thr 50 55 60 Trp Lys Ile ThrVal Pro Glu Gly Lys Val Val Val Leu Asn Phe 65 70 75 Arg Phe Ile Asp LeuGlu Ser Asp Asn Leu Cys Arg Tyr Asp Phe 80 85 90 Val Asp Val Tyr Asn GlyHis Ala Asn Gly Gln Arg Ile Gly Arg 95 100 105 Phe Cys Gly Thr Phe ArgPro Gly Ala Leu Val Ser Ser Gly Asn 110 115 120 Lys Met Met Val Gln MetIle Phe Asp Ala Asn Thr Ala Gly Asn 125 130 135 Gly Phe Met Ala Met PheSer Ala Ala Glu Pro Asn Glu Arg Gly 140 145 150 Asp Gln Tyr Cys Gly GlyLeu Leu Asp Arg Pro Ser Gly Ser Phe 155 160 165 Lys Thr Pro Asn Trp ProAsp Arg Asp Tyr Pro Ala Gly Val Thr 170 175 180 Cys Val Trp His Ile ValAla Pro Lys Asn Gln Leu Ile Glu Leu 185 190 195 Lys Phe Glu Lys Phe AspVal Glu Arg Asp Asn Tyr Cys Arg Tyr 200 205 210 Asp Tyr Val Ala Val PheAsn Gly Gly Glu Val Asn Asp Ala Arg 215 220 225 Arg Ile Gly Lys Tyr CysGly Asp Ser Pro Pro Ala Pro Ile Val 230 235 240 Ser Glu Arg Asn Glu LeuLeu Ile Gln Phe Leu Ser Asp Leu Ser 245 250 255 Leu Thr Ala Asp Gly PheIle Gly His Tyr Ile Phe Arg Pro Lys 260 265 270 Lys Leu Pro Thr Thr ThrGlu Gln Pro Val Thr Thr Thr Phe Pro 275 280 285 Val Thr Thr Gly Leu LysPro Thr Val Ala Leu Cys Gln Gln Lys 290 295 300 Cys Arg Arg Thr Gly ThrLeu Glu Gly Asn Tyr Cys Ser Ser Asp 305 310 315 Phe Val Leu Ala Gly ThrVal Ile Thr Thr Ile Thr Arg Asp Gly 320 325 330 Ser Leu His Ala Thr ValSer Ile Ile Asn Ile Tyr Lys Glu Gly 335 340 345 Asn Leu Ala Ile Gln GlnAla Gly Lys Asn Met Ser Ala Arg Leu 350 355 360 Thr Val Val Cys Lys GlnCys Pro Leu Leu Arg Arg Gly Leu Asn 365 370 375 Tyr Ile Ile Met Gly GlnVal Gly Glu Asp Gly Arg Gly Lys Ile 380 385 390 Met Pro Asn Ser Phe IleMet Met Phe Lys Thr Lys Asn Gln Lys 395 400 405 Leu Leu Asp Ala Leu LysAsn Lys Gln Cys 410 415 3 349 PRT Homo sapiens misc_feature Incyte ID No1515165 3 Met Lys Thr Leu Leu Leu Leu Leu Leu Val Leu Leu Glu Leu Gly 15 10 15 Glu Ala Gln Gly Ser Leu His Arg Val Pro Leu Arg Arg His Pro 2025 30 Ser Leu Lys Lys Lys Leu Arg Ala Arg Ser Gln Leu Ser Glu Phe 35 4045 Trp Lys Ser His Asn Leu Asp Met Ile Gln Phe Thr Glu Ser Cys 50 55 60Ser Met Asp Gln Ser Ala Lys Glu Pro Leu Ile Asn Tyr Leu Asp 65 70 75 MetGlu Tyr Phe Gly Thr Ile Ser Ile Gly Ser Pro Pro Gln Asn 80 85 90 Phe ThrVal Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro 95 100 105 Ser ValTyr Cys Thr Ser Pro Ala Cys Lys Thr His Ser Arg Phe 110 115 120 Gln ProSer Gln Ser Ser Thr Tyr Ser Gln Pro Gly Gln Ser Phe 125 130 135 Ser IleGln Tyr Gly Thr Gly Ser Leu Ser Gly Ile Ile Gly Ala 140 145 150 Asp GlnVal Ser Val Glu Gly Leu Thr Val Val Gly Gln Gln Phe 155 160 165 Gly GluSer Val Thr Glu Pro Gly Gln Thr Phe Val Asp Ala Glu 170 175 180 Phe AspGly Ile Leu Gly Leu Gly Tyr Pro Ser Leu Ala Val Gly 185 190 195 Gly ValThr Pro Val Phe Asp Asn Met Met Ala Gln Asn Leu Val 200 205 210 Asp LeuPro Met Phe Ser Val Tyr Met Ser Ser Asn Pro Glu Gly 215 220 225 Gly AlaGly Ser Glu Leu Ile Phe Gly Gly Tyr Asp His Ser His 230 235 240 Phe SerGly Ser Leu Asn Trp Val Pro Val Thr Lys Gln Ala Tyr 245 250 255 Trp GlnIle Ala Leu Asp Asn Tyr Ala Val Glu Cys Ala Asn Leu 260 265 270 Asn ValMet Pro Asp Val Thr Phe Thr Ile Asn Gly Val Pro Tyr 275 280 285 Thr LeuSer Pro Thr Ala Tyr Thr Leu Leu Asp Phe Val Asp Gly 290 295 300 Met GlnPhe Cys Ser Ser Gly Phe Gln Gly Leu Asp Ile His Pro 305 310 315 Pro AlaGly Pro Leu Trp Ile Leu Gly Asp Val Phe Ile Arg Gln 320 325 330 Phe TyrSer Val Phe Asp Arg Gly Asn Asn Arg Val Gly Leu Ala 335 340 345 Pro AlaVal Pro 4 1000 DNA Homo sapiens misc_feature Incyte ID No 456855 4ttttttcata ccatctctaa gattgctgcc gcatttgctt gttaaactga aagcatgttt 60cttgcaaagg ctctattgga aggagcagat cgaggtcttg gagaagctct tggaggcctc 120tttggaggag gtggtcagag aagagaagga ggaggaagaa atattggagg gatagttgga 180ggaattgtga attttatcag tgaggctgca gcagctcagt atactccaga accgcctccc 240actcagcagc atttcaccag tgtggaggcc tcagaaagtg aggaagttag gcgatttcgg 300caacaattta cacagctggc tggaccagac atggaggtgg gtgccactga tctgatgaat 360attctcaaca aagtcctttc taagcacaaa gatcttaaga ctgacggttt tagtcttgac 420acctgccgga gcattgtgtc tgtcatggac agtgacacga ctggtaagct gggctttgaa 480gaatttaagt atctgtggaa caacatcaag aaatggcagt gtgtttataa gcagtatgac 540agggaccatt ctgggtctct gggaagttct cagctgcggg gagctctgca ggccgcaggc 600ttccagctaa atgaacaact ttaccaaatg attgtccgcc ggtatgctaa tgaagatgga 660gatatggatt ttaacaattt catcagctgc ttggtccgcc tggatgccat gtttcgtgcc 720ttcaagtctc tggatagaga tagagatggc ctgattcaag tgtctatcaa agagtggctg 780cagttgacca tgtattcctg aagtgggaac tgagaagtca agatcctccc tggaggacag 840gactgaaaac cttgccaagc tgtacacagt tgctgatacc ctgtgcaaca gctctcattt 900cctggcaagc tctttcacaa ccctacatat ttctgatcat gtgctgcctt ttactgctga 960attaaaacag atatttcacg aaaaaaaaaa aaaaaaaaaa 1000 5 1802 DNA Homo sapiensmisc_feature Incyte ID No 947429 5 cgctgtcggt gcggcggcgc gcgtgcgggtgcaaacccga gcgtctacgc tgccatgagg 60 ggcgcgaacg cctgggcgcc actctgcctgctgctggctg ccgccaccca gctctcgcgg 120 cagcagtccc cagagagacc tgttttcacatgtggtggca ttcttactgg agagtctgga 180 tttattggca gtgaaggttt tcctggagtgtaccctccaa atagcaaatg tacttggaaa 240 atcacagttc ccgaaggaaa agtagtcgttctcaatttcc gattcataga cctcgagagt 300 gacaacctgt gccgctatga ctttgtggatgtgtacaatg gccatgccaa tggccagcgc 360 attggccgct tctgtggcac tttccggcctggagcccttg tgtccagtgg caacaagatg 420 atggtgcaga tgatttttga tgccaacacagctggcaatg gcttcatggc catgttctcc 480 gctgctgaac caaacgaaag aggggatcagtattgtggag gactccttga cagaccttcc 540 ggctctttta aaacccccaa ctggccagaccgggattacc ctgcaggagt cacttgtgtg 600 tggcacattg tagccccaaa gaatcagcttatagaattaa agtttgagaa gtttgatgtg 660 gagcgagata actactgccg atatgattatgtggctgtgt ttaatggcgg ggaagtcaac 720 gatgctagaa gaattggaaa gtattgtggtgatagtccac ctgcgccaat tgtgtctgag 780 agaaatgaac ttcttattca gtttttatcagacttaagtt taactgcaga tgggtttatt 840 ggtcactaca tattcaggcc aaaaaaactgcctacaacta cagaacagcc tgtcaccacc 900 acattccctg taaccacggg tttaaaacccaccgtggcct tgtgtcaaca aaagtgtaga 960 cggacgggga ctctggaggg caattattgttcaagtgact ttgtattagc cggcactgtt 1020 atcacaacca tcactcgcga tgggagtttgcacgccacag tctcgatcat caacatctac 1080 aaagagggaa atttggcgat tcagcaggcgggcaagaaca tgagtgccag gctgactgtc 1140 gtctgcaagc agtgccctct cctcagaagaggtctaaatt acattattat gggccaagta 1200 ggtgaagatg ggcgaggcaa aatcatgccaaacagcttta tcatgatgtt caagaccaag 1260 aatcagaagc tcctggatgc cttaaaaaataagcaatgtt aacagtgaac tgtgtccatt 1320 taagctgtat tctgccattg cctttgaaagatctatgttc tctcagtaga aaaaaaaata 1380 cttataaaat tacatattct gaaagaggattccgaaagat gggactggtt gactcttcac 1440 atgatggagg tatgaggcct ccgagatagctgagggaagt tctttgcctg ctgtcagagg 1500 agcagctatc tgattggaaa cctgccgacttagtgcggtg ataggaagct aaaagtgtca 1560 agcgttgaca gcttggaagc gtttatttatacatctctgt aaaaggatat tttagaattg 1620 agttgtgtga agatgtcaaa aaaagattttagaagtgcaa tatttatagt gttatttgtt 1680 tcaccttcaa gcctttgccc tgaggtgttacaatcttgtc ttgcgttttc taaatcaatg 1740 cttaataaaa tatttttaaa ggaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaacga 1800 at 1802 6 2073 DNA Homo sapiensmisc_feature Incyte ID No 1515165 6 cggagggggc aagggagaag ctgctggtcggactcacaat gaaaacgctc cttcttttgc 60 tgctggtgct cctggagctg ggagaggcccaaggatccct tcacagggtg cccctcagga 120 ggcatccgtc cctcaagaag aagctgcgggcacggagcca gctctctgag ttctggaaat 180 cccataattt ggacatgatc cagttcaccgagtcctgctc aatggaccag agtgccaagg 240 aacccctcat caactacttg gatatggaatacttcggcac tatctccatt ggctccccac 300 cacagaactt cactgtcatc ttcgacactggctcctccaa cctctgggtc ccctctgtgt 360 actgcactag cccagcctgc aagacgcacagcaggttcca gccttcccag tccagcacat 420 acagccagcc aggtcaatct ttctccattcagtatggaac cgggagcttg tccgggatca 480 ttggagccga ccaagtctct gtggaaggactaaccgtggt tggccagcag tttggagaaa 540 gtgtcacaga gccaggccag acctttgtggatgcagagtt tgatggaatt ctgggcctgg 600 gatacccctc cttggctgtg ggaggagtgactccagtatt tgacaacatg atggctcaga 660 acctggtgga cttgccgatg ttttctgtctacatgagcag taacccagaa ggtggtgccg 720 ggagcgagct gatttttgga ggctacgaccactcccattt ctctgggagc ctgaattggg 780 tcccagtcac caagcaagct tactggcagattgcactgga taactatgct gtggagtgtg 840 ccaaccttaa cgtcatgccg gatgtcaccttcaccattaa cggagtcccc tataccctca 900 gcccaactgc ctacacccta ctggacttcgtggatggaat gcagttctgc agcagtggct 960 ttcaaggact tgacatccac cctccagctgggcccctctg gatcctgggg gatgtcttca 1020 ttcgacagtt ttactcagtc tttgaccgtgggaataaccg tgtgggactg gccccagcag 1080 tcccctaagg aggggccttg tgtctgtgcctgcctgtctg acagaccttg aatatgttag 1140 gctggggcat tctttacacc tacaaaaagttattttccag agaatgtagc tgtttccagg 1200 gttgcaactt gaattaagac caaacagaacatgagaatac acacacacac acacatatac 1260 acacacacac acttcacaca tacacaccactcccaccacc gtcatgatgg aggaattacg 1320 ttatacattc atattttgta ttgatttttgattatgaaaa tcaaaaattt tcacatttga 1380 ttatgaaaat ctccaaacat atgcacaagcagagatcatg gtataataaa tccctttgca 1440 actccactca gccctgacaa cccatccacacacggccagg cctgtttatc tacactgctg 1500 cccactcctc tctccagctc cacatgctgtacctggatca ttctgaagca aattccgagc 1560 attacatcat tttgtccata aatatttctaacatccttaa atatacaatc ggaattcaag 1620 catctcccat tgtcccacaa atgtttggctgtttttgtag ttggattgtt tgtattagga 1680 ttcaagcaag gcccatatat tgcatttatttgaaatgtct gtaagtctct ttccatctac 1740 agagtttagc acatttgaac gttgctggttgaaatcccga ggtgtcattt gacatggttc 1800 tctgaactta tctttcctat aaaatggtagttagatctgg aggtctgatt ttgtggcaaa 1860 aatacttcct aggtggtgct gggtacttcttgttgcatcc tgtcaggagg cagataatgc 1920 tggtgcctct ctattggtaa tgttaagactgctgggtggg tttggagttc ttggctttaa 1980 tcattcatta caaagttcag cattttaaaaaaaaaaaaaa aaaaggaaaa aagaaagaaa 2040 aagagaaaaa agaaaaaaaa aaggaaagagggg 2073 7 266 PRT Homo sapiens misc_feature Incyte ID No 164403 7 MetPhe Leu Val Asn Ser Phe Leu Lys Gly Gly Gly Gly Gly Gly 1 5 10 15 GlyGly Gly Gly Gly Leu Gly Gly Gly Leu Gly Asn Val Leu Gly 20 25 30 Gly LeuIle Ser Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly GlyGly Gly Gly Gly Gly Gly Thr Ala Met Arg Ile Leu 50 55 60 Gly Gly Val IleSer Ala Ile Ser Glu Ala Ala Ala Gln Tyr Asn 65 70 75 Pro Glu Pro Pro ProPro Arg Thr His Tyr Ser Asn Ile Glu Ala 80 85 90 Asn Glu Ser Glu Glu ValArg Gln Phe Arg Arg Leu Phe Ala Gln 95 100 105 Leu Ala Gly Asp Asp MetGlu Val Ser Ala Thr Glu Leu Met Asn 110 115 120 Ile Leu Asn Lys Val ValThr Arg His Pro Asp Leu Lys Thr Asp 125 130 135 Gly Phe Gly Ile Asp ThrCys Arg Ser Met Val Ala Val Met Asp 140 145 150 Ser Asp Thr Thr Gly LysLeu Gly Phe Glu Glu Phe Lys Tyr Leu 155 160 165 Trp Asn Asn Ile Lys LysTrp Gln Ala Ile Tyr Lys Gln Phe Asp 170 175 180 Val Asp Arg Ser Gly ThrIle Gly Ser Ser Glu Leu Pro Gly Ala 185 190 195 Phe Glu Ala Ala Gly PheHis Leu Asn Glu His Leu Tyr Ser Met 200 205 210 Ile Ile Arg Arg Tyr SerAsp Glu Gly Gly Asn Met Asp Phe Asp 215 220 225 Asn Phe Ile Ser Cys LeuVal Arg Leu Asp Ala Met Phe Arg Ala 230 235 240 Phe Lys Ser Leu Asp LysAsp Gly Thr Gly Gln Ile Gln Val Asn 245 250 255 Ile Gln Glu Trp Leu GlnLeu Thr Met Tyr Ser 260 265 8 468 PRT Homo sapiens misc_feature IncyteID No 2589009 8 Met Leu Pro Ala Ala Leu Thr Ser Phe Leu Gly Pro Phe LeuLeu 1 5 10 15 Ala Trp Val Leu Pro Leu Ala Arg Gly Gln Thr Pro Asn TyrThr 20 25 30 Arg Pro Val Phe Leu Cys Gly Gly Asp Val Thr Gly Glu Ser Gly35 40 45 Tyr Val Ala Ser Glu Gly Phe Pro Asn Leu Tyr Pro Pro Asn Lys 5055 60 Lys Cys Ile Trp Thr Ile Thr Val Pro Glu Gly Gln Thr Val Ser 65 7075 Leu Ser Phe Arg Val Phe Asp Met Glu Leu His Pro Ser Cys Arg 80 85 90Tyr Asp Ala Leu Glu Val Phe Ala Gly Ser Gly Thr Ser Gly Gln 95 100 105Arg Leu Gly Arg Phe Cys Gly Thr Phe Arg Pro Ala Pro Val Val 110 115 120Ala Pro Gly Asn Gln Val Thr Leu Arg Met Thr Thr Asp Glu Gly 125 130 135Thr Gly Gly Arg Gly Phe Leu Leu Trp Tyr Ser Gly Arg Ala Thr 140 145 150Ser Gly Thr Glu His Gln Phe Cys Gly Gly Arg Met Glu Lys Ala 155 160 165Gln Gly Thr Leu Thr Thr Pro Asn Trp Pro Glu Ser Asp Tyr Pro 170 175 180Pro Gly Ile Ser Cys Ser Trp His Ile Ile Ala Pro Ser Asn Gln 185 190 195Val Ile Met Leu Thr Phe Gly Lys Phe Asp Val Glu Pro Asp Thr 200 205 210Tyr Cys Arg Tyr Asp Ser Val Ser Val Phe Asn Gly Ala Val Ser 215 220 225Asp Asp Ser Lys Arg Leu Gly Lys Phe Cys Gly Asp Lys Ala Pro 230 235 240Ser Pro Ile Ser Ser Glu Gly Asn Glu Leu Leu Val Gln Phe Val 245 250 255Ser Asp Leu Ser Val Thr Ala Asp Gly Phe Ser Ala Ser Tyr Arg 260 265 270Thr Leu Pro Arg Asp Ala Val Glu Lys Glu Ser Ala Leu Ser Pro 275 280 285Gly Glu Asp Val Gln Arg Gly Pro Gln Ser Arg Ser Asp Pro Lys 290 295 300Thr Gly Thr Gly Pro Lys Val Lys Pro Pro Thr Lys Pro Lys Ser 305 310 315Gln Pro Ala Glu Thr Pro Glu Ala Ser Pro Ala Thr Gln Ala Thr 320 325 330Pro Val Ala Pro Ala Ala Pro Ser Ile Thr Cys Pro Lys Gln Tyr 335 340 345Lys Arg Ser Gly Thr Leu Gln Ser Asn Phe Cys Ser Ser Ser Leu 350 355 360Val Val Thr Gly Thr Val Lys Thr Met Val Arg Gly Pro Gly Glu 365 370 375Gly Leu Thr Val Thr Val Ser Leu Leu Gly Val Tyr Lys Thr Gly 380 385 390Gly Leu Asp Leu Pro Ser Pro Pro Ser Gly Thr Ser Leu Lys Leu 395 400 405Tyr Val Pro Cys Arg Gln Met Pro Pro Met Lys Lys Gly Ala Ser 410 415 420Tyr Leu Leu Met Gly Gln Val Glu Glu Asn Arg Gly Pro Ile Leu 425 430 435Pro Pro Glu Ser Phe Val Val Leu Tyr Arg Ser Asn Gln Asp Gln 440 445 450Ile Leu Asn Asn Leu Ser Lys Arg Lys Cys Pro Ser Gln Pro Arg 455 460 465Thr Ala Ala 9 396 PRT Homo sapiens misc_feature Incyte ID No 181994 9Met Lys Thr Leu Leu Leu Leu Leu Leu Val Leu Leu Glu Leu Gly 1 5 10 15Glu Ala Gln Gly Ser Leu His Arg Val Pro Leu Arg Arg His Pro 20 25 30 SerLeu Lys Lys Lys Leu Arg Ala Arg Ser Gln Leu Ser Glu Phe 35 40 45 Trp LysSer His Asn Leu Asp Met Ile Gln Phe Thr Glu Ser Cys 50 55 60 Ser Met AspGln Ser Ala Lys Glu Pro Leu Ile Asn Tyr Leu Asp 65 70 75 Met Glu Tyr PheGly Thr Ile Ser Ile Gly Ser Pro Pro Gln Asn 80 85 90 Phe Thr Val Ile PheAsp Thr Gly Ser Ser Asn Leu Trp Val Pro 95 100 105 Ser Val Tyr Cys ThrSer Pro Ala Cys Lys Thr His Ser Arg Phe 110 115 120 Gln Pro Ser Gln SerSer Thr Tyr Ser Gln Pro Gly Gln Ser Phe 125 130 135 Ser Ile Gln Tyr GlyThr Gly Ser Leu Ser Gly Ile Ile Gly Ala 140 145 150 Asp Gln Val Ser ValGlu Gly Leu Thr Val Val Gly Gln Gln Phe 155 160 165 Gly Glu Ser Val ThrGlu Pro Gly Gln Thr Phe Val Asp Ala Glu 170 175 180 Phe Asp Gly Ile LeuGly Leu Gly Tyr Pro Ser Leu Ala Val Gly 185 190 195 Gly Val Thr Pro ValPhe Asp Asn Met Met Ala Gln Asn Leu Val 200 205 210 Asp Leu Pro Met PheSer Val Tyr Met Ser Ser Asn Pro Glu Gly 215 220 225 Gly Ala Gly Ser GluLeu Ile Phe Gly Gly Tyr Asp His Ser His 230 235 240 Phe Ser Gly Ser LeuAsn Trp Val Pro Val Thr Lys Gln Ala Tyr 245 250 255 Trp Gln Ile Ala LeuAsp Asn Ile Gln Val Gly Gly Thr Val Met 260 265 270 Phe Cys Ser Glu GlyCys Gln Ala Ile Val Asp Thr Gly Thr Ser 275 280 285 Leu Ile Thr Gly ProSer Asp Lys Ile Lys Gln Leu Gln Asn Ala 290 295 300 Ile Gly Ala Ala ProVal Asp Gly Glu Tyr Ala Val Glu Cys Ala 305 310 315 Asn Leu Asn Val MetPro Asp Val Thr Phe Thr Ile Asn Gly Val 320 325 330 Pro Tyr Thr Leu SerPro Thr Ala Tyr Thr Leu Leu Asp Phe Val 335 340 345 Asp Gly Met Gln PheCys Ser Ser Gly Phe Gln Gly Leu Asp Ile 350 355 360 His Pro Pro Ala GlyPro Leu Trp Ile Leu Gly Asp Val Phe Ile 365 370 375 Arg Gln Phe Tyr SerVal Phe Asp Arg Gly Asn Asn Arg Val Gly 380 385 390 Leu Ala Pro Ala ValPro 395

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-3, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-3, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1-3.2. An isolated polypeptide of claim 1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-3.
 3. An isolatedpolynucleotide encoding a polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide of claim
 2. 5. An isolatedpolynucleotide of claim 4 comprising a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:4-6.
 6. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 3. 7. A cell transformed with a recombinantpolynucleotide of claim
 6. 8. A transgenic organism comprising arecombinant polynucleotide of claim
 6. 9. A method of producing apolypeptide of claim 1, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. A method of claim 9, wherein thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-3.
 11. An isolated antibody which specificallybinds to a polypeptide of claim
 1. 12. An isolated polynucleotideselected from the group consisting of: a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:4-6, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence selected from the group consisting of SEQ ID NO:4-6, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodof detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:1-3.
 19. Amethod for treating a disease or condition associated with decreasedexpression of functional HPRM, comprising administering to a patient inneed of such treatment the composition of claim
 17. 20. A method ofscreening a compound for effectiveness as an agonist of a polypeptide ofclaim 1, the method comprising: a) exposing a sample comprising apolypeptide of claim 1 to a compound, and b) detecting agonist activityin the sample.
 21. A composition comprising an agonist compoundidentified by a method of claim 20 and a pharmaceutically acceptableexcipient.
 22. A method for treating a disease or condition associatedwith decreased expression of functional HPRM, comprising administeringto a patient in need of such treatment a composition of claim
 21. 23. Amethod of screening a compound for effectiveness as an antagonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingantagonist activity in the sample.
 24. A composition comprising anantagonist compound identified by a method of claim 23 and apharmaceutically acceptable excipient.
 25. A method for treating adisease or condition associated with overexpression of functional HPRM,comprising administering to a patient in need of such treatment acomposition of claim
 24. 26. A method of screening for a compound thatspecifically binds to the polypeptide of claim 1, the method comprising:a) combining the polypeptide of claim 1 with at least one test compoundunder suitable conditions, and b) detecting binding of the polypeptideof claim 1 to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide of claim
 1. 27. A method ofscreening for a compound that modulates the activity of the polypeptideof claim 1, the method comprising: a) combining the polypeptide of claim1 with at least one test compound under conditions permissive for theactivity of the polypeptide of claim 1, b) assessing the activity of thepolypeptide of claim 1 in the presence of the test compound, and c)comparing the activity of the polypeptide of claim 1 in the presence ofthe test compound with the activity of the polypeptide of claim 1 in theabsence of the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Amethod for a diagnostic test for a condition or disease associated withthe expression of HPRM in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HPRM in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, further comprising a label.
 35. A method ofdiagnosing a condition or disease associated with the expression of HPRMin a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 1-3, or an immunogenic fragment thereof, under conditionsto elicit an antibody response, b) isolating antibodies from the animal,and c) screening the isolated antibodies with the polypeptide, therebyidentifying a polyclonal antibody which specifically binds to apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-3.
 37. A polyclonal antibody produced by amethod of claim
 36. 38. A composition comprising the polyclonal antibodyof claim 37 and a suitable carrier.
 39. A method of making a monoclonalantibody with the specificity of the antibody of claim 11, the methodcomprising: a) immunizing an animal with a polypeptide consisting of anamino acid sequence selected from the group consisting of SEQ ID NO:1-3, or an immunogenic fragment thereof, under conditions to elicit anantibody response, b) isolating antibody producing cells from theanimal, c) fusing the antibody producing cells with immortalized cellsto form monoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich specifically binds to a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1-3.
 40. Amonoclonal antibody produced by a method of claim
 39. 41. A compositioncomprising the monoclonal antibody of claim 40 and a suitable carrier.42. The antibody of claim 11, wherein the antibody is produced byscreening a Fab expression library.
 43. The antibody of claim 11,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-3 in a sample, the method comprising: a) incubating theantibody of claim 11 with the sample under conditions to allow specificbinding of the antibody and the polypeptide, and b) detecting specificbinding, wherein specific binding indicates the presence of apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-3 in the sample.
 45. A method of purifying apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-3 from a sample, the method comprising: a)incubating the antibody of claim 11 with the sample under conditions toallow specific binding of the antibody and the polypeptide, and b)separating the antibody from the sample and obtaining the purifiedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-3.
 46. A microarray wherein at least oneelement of the microarray is a polynucleotide of claim
 13. 47. A methodof generating an expression profile of a sample which containspolynucleotides, the method comprising: a) labeling the polynucleotidesof the sample, b) contacting the elements of the microarray of claim 46with the labeled polynucleotides of the sample under conditions suitablefor the formation of a hybridization complex, and c) quantifying theexpression of the polynucleotides in the sample.
 48. An array comprisingdifferent nucleotide molecules affixed in distinct physical locations ona solid substrate, wherein at least one of said nucleotide moleculescomprises a first oligonucleotide or polynucleotide sequencespecifically hybridizable with at least 30 contiguous nucleotides of atarget polynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:2.
 58. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:3.
 59. A polynucleotideof claim 12, comprising the polynucleotide sequence of SEQ ID NO:4. 60.A polynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:5.
 61. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:6.